FI120452B - Colchicine derivatives and their therapeutic use - Google Patents

Abstract

The present invention relates to novel colchicine derivatives having antiproliferative, antitumoral and antiinflammatory activities. They can be included in pharmaceutical formulations useful for the parenteral, oral and topical administrations.

Description

5

Kolkisiim derivatives and their therapeutic use Kolkidnderivat ooh deras terapeutiska användnitig

The present invention relates to novel colchicine derivatives having increased anti-tumor, anti-tumor and anti-inflammatory effects, processes for their preparation and pharmaceutical preparations containing them. Colchicine is a 1.Ö known pseudo-alkaloid that has been used for a very long time to treat gout .; it works liv via fast 'and Specifically, even if it should only be used for short periods of time. because of its toxicity. in addition, colchicine is a very potent antibacterial agent *. the effect of which is related to a blocking mechanism of mitotic winding in cell division; these latter 15 features have been thoroughly investigated to determine the potential antitumor effect and many colchicine derivatives have been prepared for this purpose.

Triceps as such, and many of its derivatives, have not been used, in terms of the hazard / benefit ratio, due to their considerable toxicity. Only '20 of one colchicine derivative, denTefcolkin, is used to some extent in the treatment of cancers in the treatment of cancer. As for use in the control of inflammation, the only triglyceride derivative available on the market is a thiocolchicoside comprising a thiomethyl group on Gp and a hydroxyl present on the glucose molecule; therapeutic uses of this derivative, related to lilac 25 relaxing and anti-inflammatory and antipyretic effects. The products according to the invention differ from the state of the art products in their high cure rates. In the field of antitumor research, researchers have focused their research on products that, in addition to normal cytotoxicity, have cytotoxicity against common parental cell line drug resistant strains.

30

The derivatives of the present invention have the following general formula .1: 2 -γ5 γ

Γ 7 / V

OCK. (/ Κ ~ Λ XMe 10 where: X may be Oxygen or N; 15 R 1 and R 2: which may be the same or different are linear or branched alkyl groups having from 1 to 6 carbon atoms or cycloal% superfamily; or Saturated or unsaturated acyl groups having from 16 to 22 carbon atoms or a BD glucose residue as such or a B * D glucose unit in which the hydroxyls of the 4- and O-positions are protected by ketal aliphatic or aromatic or heximromic aldehyde; A -R 4 group having a methylene group bonded to the 5-position carbon and a methine group having the same absorptive configuration as the colchicine is bonded to the tropolone ring, or -CKCl 4 OR 4 methylene having the methylene group bonded to the 5-position carbon and the tropolone ring is an absolute S configuration; R3 is an acyl group containing from 2 to 6 carbon atoms having 1 to 3 halogen atoms, preferably fluorine or chlorine, or a natural amino acid. the amino group may be a free or protected acyl group derived from trifluoroacetamide or benzamide; R4 is an acyl residue of a dicarboxylic acid having 4 to 6 carbon atoms or an acyl residue of a natural amino acid in which the amino group may be free or protected to trifluoroacetamide or benzamide, or a glycoside residue consisting of D-glucose-3, P-galacto, L-ramnooslsta. In this case, the sugars have a chemotaxis effect on melanocytes, hepatocytes, fibroblasts or derive a derivative to the northwest, Preferred compounds of formula I are those wherein X is stitch,

R1 and R2, which are the same or different, are preferably hydrogen or C1-4alkyl.

Y is preferably a group of the formula -CH2CH-CH-NH-R6 as defined above.

.10

The starting materials for the preparation of the products according to the invention are the natural substances colchicine (1; X = O; R 1 = R 2 -Me: Y 'CH 2 CH-NHAc), 2- (Meryllyl-colchicine (1; X-O; R 1 -); M; R 2 - Me; Y (R) -CH-NHAc), 3-O-demethylcholizine (1; X = O; R (= Me; R 2 - H; Y 1 R 2 -CH-NHAc), colchicoside (1; X = 15 °; R 1 ™ Me; R 2 'β-Ρ-glucose; Y = · CH 2 -CH-NHAe) obtained from plant materials by methods known in the literature. These natural substances are obtained by treatment with methyl mercaptan in a basic solution in the same manner according to methods known in the literature for use of thio derivatives as syntheses for the preparation of derivatives of general formula 1 wherein X is sulfur

The starting materials used for the preparation of derivatives of general formula 1 wherein R 1 and R 2 are alkyl or acyl groups are colchicine or thiocolchicine syntheses with methyl at the 2- or 3-position. These chthones are alkylated or acylated using well-known methods to obtain the phenol derivative. Similarly, derivatives of general formula 1 wherein R a or R 2 is a BD glucose residue or an hD glucose residue in which the hydroxyls of the 4- and 6-positions are protected by ketals with aliphatic or aromatic aldehydes are prepared from colchicine or thiocolchic these 30 · synthons are reacted with α-bromo-tetm-acetyl-1D-glucose or 2,3-di-δ-dichloroacetyl-BD-glucose containing a subgroup of hydroxyls at the 4- and 6-positions (see Canadian Patent Publication 956939). After deprotection of the 4 protecting acyl groups by methods described in the literature, 2- or 3-position glycosylated derivatives of the present invention are obtained; Derivatives of Formula 1 wherein Y is -CH 2 -GSIrNH-R 8 -hyd., Are prepared by subjecting acid-catalyzed N-deacetylation moieties of the methoxy · or thioalkysulfuric acid syntheses at the 2- and 3-positions followed by acylation of the primary amino group. -3 as an acid reactive derivative containing fluorine or chlorine free radicals, or a natural amino acid which may have a free or amino protected amino group or a trifluoroacetate or a benzamide. In this way the formula .1. derivatives with 1 & $. is as defined above. Derivatives of Formula 1: wherein Y is a -CH-GH3-O2R4 group are obtained by subjecting the colchicine or thiol-chromium sulfinones containing methoxy or thyloxy group at the 2 and 3 positions to N-deacetylation followed by treatment with sodium nitrate and acetic acid depletion. . 15 The cloheptane ring disappears to form syntheses of formula ί, with Y -GH-GH ^ OH. (J. Med. Ghent .: 36, 544, 1993), The resulting primary alcohol group produces a reaction state of suitably activated dicarboxylic acids or activated natural amino acids which may be free or protected to trifluoroacetamide or benzamide, or D-glucose, D-galactose, , With a reactive form of L-20 fucose or L-rhamnose sugars, derivatives of general formula I wherein Y-CH-CH 2 R 4, wherein R 4 is as defined above,

The following table shows the antimitotic activity of some of the derivatives of the invention on tumor cell lines. Taxol and colchicine have been used as reference substances.

5

Table In vitro cytotoxic activity of some novel cochlecin derivatives

Agents IC5p (nM) 5 Cellular

Egg-Thick- Vas.cap.

series of intestine Breast lung cells.

Taxol 6.1 3.5 3.2 1.7 299

Colchicine 5.3 3.9 4.4.16 112

Compound YL 4.2 2.6 6.2 8 26

Compound HZ 9.4 1.8 5.0 19 11

Compound IVZ 6.1. 2.2 4.1 12 7 15___ This table demonstrates that Which of the new derivatives have significant advantages over resistant refractory lines, which are the major target of cyophoxic drugs. The products of the present invention may be incorporated into pharmaceutical formulations useful for the administration of this drug. Formulations for parenteral, oral, transdermal, or topical administration may be prepared in a starch-specific manner.

Of the yeast excipients that can be used to prepare these formulations, natural and synthetic phosphblipids have proven to be particularly useful in the preparation of a liposome form for the parenteral, transdermal, or topical route of administration; the latter two preparations being particularly useful for treating inflammatory conditions in joints or peripheral veins; these preparations also being useful for the topical treatment of epithelial tumors of the skin and of conditions of skin hyperplasia such as psoriasis. Particularly useful in the specific control of tumors, besides the phospholipids which enable the drug to be cleaved in the jspome form, some surfactants, such as polyethoxylated 6 castor oil, such as Crernoform L.50 or polysorbate, such as Tween, which act synergistically with the active ingredient have proved to be useful. In Oncology What products are used at a dose of 1 ...... 1 000 mg / in2, while in the fight against inflammation the dose ranges from 1 to 20 mg per unit dose, one to five or more times daily, All pharmaceuticals such as ampoules, capsules, ointments and the like can be prepared from most of these derivatives.

The following examples further illustrate the invention.

10

Example 1 - Preparation of M-deacetyl-N-trifluoroacetyl-B0-demethyl-1-yl-cyclopentyl-thiocolchicine or Compound ((1; X-S; R1-Me; R2-C5H9; Y-CH2CeM11I-COCF3) 15 g 3-O-Demethylthiothiolcolicin (1; X-S; R 1 -Me; R 2 ™ H; Y · -GH 2 ClliyHAcl) is dissolved in 300% 20% sulfuric acid and treated at 100 ° C under nitrogen for 36 hours. The reaction mixture is neutralized to give 12 g N-Deacetyl-B0-demethyl-thiocolchicine (1; X = S; Ra-Me; R2-H; Y ™ CH2CHNH2) This product is dissolved in acetone and reacted with 3 equivalents of trifluoroacetic anhydride while stirring vigorously with anhydrous Na2CO3. in the presence of ... The reaction mixture is filtered after f hours and the solution is evaporated to dryness The residue consisting of SO-demethyl-MiS-O-bistrifluoroacetylation-colchicine is hydrolyzed in methanol containing N 2 Cl and the residue is evaporated to dryness in vacuo. is mixed with acetone the solution is filtered and refluxed at 8 nm with 5 equivalents of cyanopentyl bromide in the presence of sodium carbonate. The salts are filtered off, the solution is evaporated to dryness and the residue is purified by chromatography on a silica gel column using ethyl acetate as the eluent. The product is obtained by crystallization from 8.6 g of a mixture of acetone and hexane, M in / z 523.

30 7

Example II-Preparation of N-Acetyl-N-tilylacetyl-acetyl-S-N, N-demethyl-S-N-isopropyl-ylcoliquimim, i.e. Compound IIZ (1; X-S; R1-Me; R2-iPr; Y-CH2-CH NH-COCF3). ) To prepare this derivative, the procedure described in Example 1 is repeated except that the reagent is isopentyl bromide and not cyclopentyl bromide. After purification of the crude reaction product on silica gel and crystallization, 7.6 g of product, M't-a ml.z 497, are obtained.

Example 10 Preparation of HI-N-deacetyl-N-triilionacetyl thioalkosicide or compound ΙΙΪΖ (1; X-S; R {- Me; R 2 = β-D-glucosyl; Y-CH 2 -CH-NHGOOCF 3) 10 g of K-deacetyl ecosilicosHa (1; XS; R [= Me; R2 ™ ikD-glucose; Y -CH2-GH-NH4]) is dissolved in acetone and treated for 2 hours at 10 ° C. with three 15 equivalents of trifluoroacetic anhydride. The mixture is evaporated to dryness and the residue is crystallized from isopropanol and then into ethanol. 8.5 g of product are obtained, M'fa m / z 617.

Example IV - Preparation of NYN-Trifluoroacetyl-α-phenylglycyl-deacyl-yl-colimic, i.e., Compound IVZ (1; X = S; R 5 * R 2 = · Me; Y 1 -C 3 H -NH-CO-20GHNNHCOCF 3) Ph}.

400 mg of N-deacetyl "thiocolchicine (1; X-S; R 1 R 2 Me; Y-C 1 H 2 -GTFNI:?) (1.07 mmol) and 265 µg (1.07 mmol) of LN-tert-butyl-acetyl-α -phenylglycols are dissolved together with K) any methylene chloride · in a nitrogen atmosphere, 221 mg (1.07 mmol) of Ν, Ν-dicyclohexylcarbodiimide are added to this solution while stirring until the reagents are lost, and the reaction mixture is cooled to -30 ° C and The chloromethylene solution is concentrated and purified by filtration on silica gel, eluting with a mixture of methylene chloride and methanol 98: 2 After crystallization from a mixture of methylene chloride and ethyl ether, 30 mg of the product, 8 - (M-iii fl «oriasety yti ~ L ~ aIaiiy y 1 i) 'Preparation of deasety y 1 spore f ki si ί π in el 1 y idisie VZ (I; X - S; R | - R2 ~ Me, Y - CH2-NHCD-CH (NHeOCB3) CXI%) 5 400 mg of N-deacetylthiocolchicine (1.07 mmol) are reacted with N cetyl-Lralani.iniii., one-equimolecite, and one equivalent of N, N-dicycloydoxyl-4-diphosphide in 10 ml of methylene chloride and under a phosphatic atmosphere until the reagents have disappeared. The reaction mixture is cooled to -30 ° C and filtered to remove the precipitated urea: Kionm: ethylene is concentrated and purified by filtration through silica gel, eluting with a 98: 2 mixture of methylene chloride and methanol, to give methylene chloride and ethyl acetate. mg product, M + am / z 540,

Example VI - Preparation of N- (1S- (2-trifluoromethyl) -methylmethyl) -hazeacyl-free, i.e. Compound VIZ (1; X-S; R 1 = R 2 -Me; Y-CM 2 -CH-MHCO-GH (NHCOCP 3) CH 2 -CHrSMe),

The procedure of Example IV is repeated, reacting the N-MCI fluoroacetylmethyl. From 400 mg of N-deacetylthiocolchicine, 84 mg of product are obtained by fractional crystallization and purification of the reaction residue chronographically, m / z 600.

Example VII - Preparation of N- (o-Phenylglycyl) 1-Deacetyl-3-Phenol, i.e. Compound VHZ (1; X-S; R; i-%-Me; Y ** ·· (X: 12 -CH-MHCO -GeNH2 - Ph).

400 mg of the product obtained in Example IV are dissolved in 5 g of 50% acetone under conditions containing 120 µg of potassium carbonate, and the mixture is heated at 60 ° C with stirring for 5 hours, the reaction mixture is cooled, saturated with NaCl and extracted with chloroform. The organic phase is dried over anhydrous sodium sulfate, then concentrated to dryness and the residue is chromatographed on silica gel using a 98: 2 → methylene chloride / methanol mixture. 160 mg of product are obtained. M'®'a m / z 506.

..9

Example: Preparation of Vil - N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N, N; CDlCH 2 -CH 2 -CH-CH-1 CH 3 -CH 3 -CH 2 -CH 2 -Ci-NH-C (> F 3).

500 mg of N-deacetyl "N'-trifluoroacetyl -3 - () - demethylthiocoicamers (1; X-S; R 1 -Me; R 2 -H; Y C 6 -C 6 -CH-NHC.OCFa) are dissolved in 2.5 mL of pyridime. and to it is added 500 mg of ximemimeppe chloride at 0 [deg.] C. The reaction solution is allowed to stand overnight at room temperature then poured onto ice The resulting precipitate is separated and crystallized from a mixture of acetone and hexane, M + am / z 715.

10

Example IX "5,6" Dihydro-6 (S) ~ [(β-D-glucopyraliosyloxymethyl] -1,2,3 tri-nethoxy-9- (methylthio) -8H-cycloheptaia] naphthalen-8-one that is, preparation of 1KZ (1; X-S; R, ~ 1¾ ~ Me; Y = * CH-C% Ä * P-g / mole)) 15 g of deacetylthiocolchicine are treated with sodium nitrate to give 4 g of 5,6-dihydro- 6 (: SX (hydroxyacetyl) ··· 1,2,3 ”trimethyl) xy-9-methylthio-8H-cyclohalta (naphthalen-8-one) following the procedure described in J. Med. ., Ms 544, 1993 The resultant product and 26 g of α-bromotetraacetyl glucose are treated together for 12 hours under reflux in acetonitrile, under conditions of 85 g of mercuric (II) cyanide. The salts are filtered off and the solution is evaporated to dryness. is mixed with 70% acetone and treated for two hours with 15% sodium hydrate, and the mixture is neutralized, extracted with ethyl acetate and chromatographed on silica gel eluting with methylene chloride and ethanol. With an Ori mixture, 2.1 g of product is obtained, M + a m / z. 536.

. 25

Example X Preparation of 5,6-Dihydro-6- (S) - (hydroxymethyl) -1,2,3 "trimethoxy-9- (methylthio) -8H" cyclohepta [a] lalalt-8-one succinyl ester, Compound XX ( 1; XS; Rs =% = Me; Y - t: B-CH2 O0C0CII2Cl (i2CO2H).

1.0 g of N-deacetylthiocolch line are treated as described in Example IX. The resulting naphthalene-X-om is dissolved in pyridine and treated with an excess of succinic anhydride for a little over 24 hours. The reaction mixture is cooled, poured into 10 volumes of water and extracted with methylene chloride. The organic phase is concentrated to a small volume and purified on silica gel eluted with methylene chloride, water and. 70: 30: 5 methanol. After crystallization from methanol, 7 g of product are obtained, M + a / n 474.

5

Claims (8)

11 Compounds of formula 1: λ XHe 5 1 wherein: X may be an oxygen or sulfur atom; R 1 and R 2, which may be the same or different, are straight or branched chain alkyl groups or cycloalkyl groups of 1 to 6 carbon atoms; or saturated or unsaturated acyl groups containing from 16 to 22 carbon atoms or a β-D-glucose moiety as such or a β-D-glucose moiety wherein the hydrolysates of the 4- and 6-positions are protected by ketalic aliphatic or aromatic or heteroaromatic aldehydes; Y is a -CH 2 -CH-NH-R 3 group having a methylene group bonded to the 5-position carbon, and a methine group having the same absolute configuration as the colchicine bound to the tropoly ring; R3 is an acyl group containing from 2 to 6 carbon atoms having from 1 to 3 halogen atoms, preferably fluorine or chlorine, or from a natural amino acid where the amino group may be free or protected to trifluoroacetamide or benzamide, provided that when R1 and R2 is methyl, then X is an oxygen or sulfur atom, R1 is not -COCH2F, -COCH2Cl, -COCH2Br, -COCH2I; when R 1 and R 2 are methyl, X is an oxygen atom, R 3 is not -COCCl 3, -COCHCl 2, -COCHF 2, -COCH 2 CHCl 1 CH 3; -COCH2CH2CHBrCH3. Compounds according to claim 1, characterized in that X is sulfur. Compounds according to claim 1, characterized in that X is oxygen. 10 Compounds according to claim 2 or 3, characterized in that R 1 and R 2, which are the same or different, are straight or branched chain alkyl groups or cycloalkyl groups containing from 1 to 6 carbon atoms. Compounds according to any one of the preceding claims, characterized in that Y is a group of the formula -CH 2 -CH-NH-R 3 as defined in claim 1. 20 A process for the preparation of the compounds according to claims 1-5, wherein the colchicine, 2-O-demethylcholcicin, 3-O-demethylcholysicin, colchicosin are subjected to one or more of the following reactions: a) reaction with 1mercapolar in a basic solution; b) alkylation, acylation or glycosylation of phenol groups; c) N-deacetylation by acid catalysis followed by acylation of the primary amino function with R 3 -COOH or a derivative thereof, wherein R 3 is as defined in claim 1. 30 13 Pharmaceutical compositions containing as active ingredient a compound according to claims 1 to 5 in admixture with a suitable carrier, in particular natural or synthetic phospholipids. Use of compounds according to claims 1 to 5 for the preparation of medicaments having anti-proliferative, anti-tumor and anti-inflammatory effects. 14